Human PD-L1/B7-H1 Antibody

Detection of Human PD-L1/B7-H1 by Western Blot. Western blot shows lysates of human placenta tissue. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human PD-L1/B7-H1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF156) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for PD-L1/B7-H1 at approximately 50-55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

PD-L1/B7-H1 in Human Colon and Colon Cancer Tissue. PD-L1/B7-H1 was detected in immersion fixed paraffin-embedded sections of normal human colon (left panel) and human colon cancer tissue (right panel) using Goat Anti-Human PD-L1/B7-H1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF156) at 5 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to cell membranes and cytoplasm. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Human PD-L1/B7-H1 ELISA Standard Curve. Recombinant Human PD-L1/B7-H1 protein was serially diluted 2-fold and captured by Mouse Anti-Human PD-L1/B7-H1 Monoclonal Antibody (Catalog # MAB1561R) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Human PD-L1/B7-H1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF156) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998) followed by Substrate Solution (Catalog # DY999) and stopping the enzymatic reaction with Stop Solution (Catalog # DY994).

Detection of Human PD-L1/B7-H1 by Simple Western^TM. Simple Western lane view shows lysates of HDLM-2 human Hodgkin's lymphoma cell line, loaded at 0.2 mg/mL. A specific band was detected for PD-L1/B7-H1 at approximately 72 kDa (as indicated) using 50 µg/mL of Goat Anti-Human PD-L1/B7-H1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF156) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Chinese hamster PD-L1 by Western Blot In vitro and ex vivo inhibition of PD-1/PD-L1 interaction.a PD-1/PD-L1 homogenous time-resolved fluorescence (HTRF) assay. b PD-L1/PD-L1 HTRF dimerization assay. c PD-1/PD-L1 NFAT reporter bioassay. d CMV recall assay: IFN gamma production measured by ELISA following stimulation of PBMCs with CMV antigens. Source data are provided as a Source data file. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33619272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Chinese hamster PD-L1 by Western Blot Effect of compound A on ex vivo HBV-specific immune responses.a PBMCs stimulated with compound A for 1 h, followed by flow cytometric measurement of cell surface PD-L1 percentage. b PBMCs cultured in triplicate wells with HBV peptides for 10 days and T cell proliferation (thymidine incorporation) and IFN gamma secretion (ELISA) measured in the presence of compounds and antibodies. c PBMCs stimulated with compounds or antibodies overnight followed by IFN gamma producing cells measured by Fluorospot. d PBMCs collected from HBV vaccinated individuals (n = 3) on day 7 post booster vaccination were incubated with either rHBsAg alone (1 µg/ml) or rHBsAg plus compound A at 0.5 µM concentration for 5 days. Cells were then collected and stained for HBsAg-specific B cells or plasmablasts (PBs), using Atto488-labeled rHBsAg by flow cytometry. The error bars represent the standard error of mean of three individuals. statistical significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) was determined by unpaired two-tailed Student’s t test. Source data are provided as a Source data file. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33619272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human PD-L1 by ELISA Modulation of soluble co-signaling molecules in kidney-transplanted patients over time.The levels of the soluble co-signaling molecules CD30, CD40, CD137, CD40L, PD-1 and PD-L1 were assayed by ELISA in serum samples of healthy controls (n = 25) and kidney-transplanted patients (n = 59) obtained at different times: just before transplantation, and 15 days, 3 months and 1 year after transplantation. Data are shown as box-plots, in which the horizontal line within each box represents the median, the bottom and top of each box represent the 25th and 75th percentiles, the bars represent the 10th and 90th percentiles and circles indicate outliers. Unpaired and paired Wilcoxon tests were used to compare distributions between independent and dependent groups, respectively. * indicates statistically significant differences between healthy controls and kidney-transplanted patient samples, and † indicates statistically significant differences between patients samples obtained at different pre- and post-transplantation times. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25478957), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Chinese hamster PD-L1 by Western Blot Mechanism of action of compound A.a PD-L1 aAPC/CHO-K1 cells were treated with 0–500 nM compound A or compound B. PD-L1 protein visualized by native gel electrophoresis. b Native gel electrophoresis of PD-L1 protein following pre- and post-lysate treatment with compound A. c PD-L1 aAPC/CHO-K1 cell line was treated with compound A, alpha PD-1/ alpha PD-L1 antibodies, or no treatment followed by cell surface PD-L1 detected by flow cytometry. Values inside histogram represent GeoMean fluorescence intensity. d CHO-K1 cells transfected with cMyc-PD-L1 and labeled with anti-cMyc Alexa Fluor 488-conjugated antibody followed by incubation with compound A. The confocal fluorescence microscopy detects PD-L1-cMyc (green) and nucleus (blue). Images are representative of three experiments and a minimum of 50 cells observed in each experiment. e Whole-cell extracts of CHO-K1 cells co-transfected with cMyc-PD-L1 and Flag-PD-L1 were incubated with media + 0.5% DMSO, inactive (cmpd B) or active (cmpd A) compound, and subjected to immunoprecipitation (IP) with anti-cMyc magnetic beads and immunoblot with anti-Flag antibody. Blots are representative of three experiments. f PD-L1 aAPC/CHO-K1 cell line treated with compound A to allow for loss of cell surface PD-L1, followed by daily media washes. Black line represents diminishing concentration of compound A as measured by LC-MS. Red line represents reconstitution of cell surface PD-L1 as measured by flow cytometry of ≥10,000 cells per time point. g Compound A wash off and PD-L1 cell surface reconstitution in the presence of transcriptional inhibitor (actinomycin D) and protein transport inhibitor (golgi plug). Inactive compound B (gray line) referenced as negative control. Source data are provided as a Source data file. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33619272), licensed under a CC-BY license. Not internally tested by R&D Systems.